The present invention relates to nucleic acid analogs having a chelation functionality, to their uses in assay procedures, to methods of capturing them to solid supports and to methods of concentrating solutions of them.
Nucleic acid analogs having important new utilities in assay procedures and in the field of diagnostics have been described in WO 92/20703. These nucleic acid analogs had a number of new properties making them of special importance in the field of diagnostics as well as in the field of antisense therapeutics.
They typically feature a polyamide backbone bearing a sequence of ligands which are nucleic acid bases. The analogs described there have the property of hybridizing with great specificity and stability to natural nucleic acids of complementary sequence.
In order to aid the detection and the manipulation of such nucleic acid analogs in diagnostics or other assay procedures and the like operations, it is desirable to provide the nucleic acid analogs with detectable labels. It is also desirable to find ways of capturing said nucleic acid analogs on solid supports. Various labels are described in WO 92/20703. Also, the capture of the nucleic acid analogs to solid supports via bound nucleic acid or nucleic acid analog sequences acting as capture probes is described.
However, it is desirable to find alternative capture methods and in particular methods which do not require a tailored capture probe which is sequence specific but rather are generally applicable to such nucleic acid analogs.
In EP-A-0 097 373 the synthesis of nucleic acids labeled with a complexing agent is described. However, the synthesis of these compounds appears to be complicated.
Furthermore, whilst natural nucleic acids are readily and routinely concentrated by precipitation from solution by ethanol, centrifugation and resuspension, no such convenient method presently exists to aid those working with these nucleic acid analogs.
The present invention now provides according to a first aspect thereof a nucleic acid analog comprising a polymeric strand which includes a sequence of ligands bound to a backbone made up of linked backbone moieties, which analog is capable of hybridization to a nucleic acid of complementary sequence, further comprising, preferably at one terminus of said backbone a chelating moiety capable of binding at least one metal ion by chelation.
Preferably, the backbone is a polyamide, polythioamide, polysulphinamide or polysulphonamide backbone and preferably said chelating moiety is present at the N-terminus.
The chelating moiety preferably comprises a sequence of peptide bonded amino acids.
Preferred sequences of amino acids for use as chelating moieties are -His, Gly,Asp or -(His)n, where n=3 to 10, e.g. 5 or 6. The longer sequences may bind more than one metal ion per molecule of nucleic acid analog.
Alternatively, said chelating moiety may be a polycarboxylic acid substituted amine such as ethylenediamine-tetraacetic acid (EDTA) or aminotriacetic acid (NtA) and the like.
The nucleic acid analog is preferably capable of hybridizing to a nucleic acid of complementary sequence to form a hybrid which is more stable against denaturation by heat than a hybrid between the conventional deoxyribonucleotide corresponding in sequence to said analog and said nucleic acid.
Said nucleic acid analog is preferably a peptide nucleic acid in which said backbone is a polyamide backbone, each said ligand being bonded directly or indirectly to a nitrogen atom in said backbone, and said ligand bearing nitrogen atoms mainly being separated from one another in said backbone by from 4 to 8 intervening atoms.
The analog is preferably capable of hybridizing to a double stranded nucleic acid in which one strand has a sequence complementary to said analog, in such a way as to displace the other strand from said one strand.
More preferred PNA compounds for use in the invention have the formula: 
n is at least 2,
each of L1-Ln is independently selected from the group consisting of hydrogen, hydroxy, (C1-C4)alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups, heterocyclic moieties, reporter ligands and chelating moieties;
each of C1-Cn is (CR6R7)y (preferably CR6R7, CHR6CHR7 or CR6R7CH2) where R6 is hydrogen and R7 is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R6 and R7 are independently selected from the group consisting of hydrogen, (C2-C6)alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C1-C6)alkoxy, (C1-C6)aIkylthio, NR3R4 and SR5, where R3 and R4 are as described below, and R5 is hydrogen, (C1-C6)alkyl, hydroxy, alkoxy, or alkylthio-substituted (C1 to C6)alkyl or R6 and R7 taken together complete an alicyclic or heterocyclic system;
each of D1-Dn is (CR6R7)z (preferably CR6R7, CHR6CHR7 or CH2CR6R7) where R6 and R7 are as defined above;
each of y and z is zero or an integer from 1 to 10, the sum y+Z being at least 2, preferably greater than 2, but not more than 10;
each of G1-Gn-1 is xe2x80x94NR3COxe2x80x94, xe2x80x94NR3C5xe2x80x94, xe2x80x94NR3SOxe2x80x94 or xe2x80x94NR3SO2xe2x80x94, in other orientation, where R3 is as defined below;
each of A1-An and B1-Bn are selected such that:
(a) A is a group of formula (lla), (llb), (llc) or (lld), and B is N or R3N+; or
(b) A is a group of formula (lld) and B is CH; 
wherein:
X is O, S, Se, NR3, CH2 or C(CH3)2;
Y is a single bond, O, S or NR4;
each of p and q is zero or an integer from 1 to 5, the sum p+q being not more than 10;
each of r and s is zero or an integer from 1 to 5, the sum r+s being not more than 10;
each R1 and R2 is independently selected from the group consisting of hydrogen, (C1-C4)alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen; and
each R3 and R4 is independently selected from the group consisting of hydrogen, (C1-C4)alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C1-C4)alkyl, hydroxy, alkoxy, alkylthio and amino;
Q is xe2x80x94CO2H, xe2x80x94CONRxe2x80x2Rxe2x80x3, xe2x80x94SO3H or xe2x80x94SO2xe2x80x94NRxe2x80x2Rxe2x80x3 or an activated derivative of xe2x80x94CO2H or xe2x80x94SO3H; and
I is xe2x80x94NRxe2x80x2Rxe2x80x3xe2x80x2 where Rxe2x80x2 and Rxe2x80x3 are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, nucleosides, nucleotides, nucleotide diphosphates, nucleotide triphosphates, oligonucleotides, including both oligoribonucleotides and oligodeoxyribonucleotides, oligonucleosides and soluble and non-soluble polymers, and xe2x80x94Rxe2x80x3xe2x80x2 is a chelating moiety. xe2x80x9cOligonucleosidesxe2x80x9d includes nucleobases bonded to ribose and connected via a backbone other than the normal phosphate backbone of nucleic acids.
In the above structures wherein Rxe2x80x2 or Rxe2x80x3 is an oligonucleotide or oligonucleoside, such structures can be considered chimeric structures between PNA compounds and the oligonucleotide or oligonucleoside.
Generally, at least one of L1-Ln will be a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, or a nucleobase binding group.
Preferred PNA-containing compounds useful to effect binding to RNA, ssDNA and dsDNA and to form triplexing structures are compounds of the formula III, IV or V: 
wherein:
each L is independently selected from the group consisting of hydrogen, phenyl, heterocyclic moieties, naturally occurring nucleobases, and non-naturally occurring nucleobases;
each R7 is independently selected from the group consisting of hydrogen and the side chains of naturally occurring alpha amino acids;
n is an integer greater than 1,
each k, l, and m is, independently, zero or an integer from 1 to 5;
each p is zero or 1;
Rh is OH, NH2 or xe2x80x94NHLysNH2; and
Rxe2x80x2 is a chelating moiety.
The invention includes according to a second aspect thereof a method of capturing a nucleic acid analog of the kind described above, which method comprises exposing the nucleic acid analog to a solid support bearing chelatable metal ions bonded thereto under conditions such that the chelating moiety of the nucleic acid analog chelates the said bound metal ions, so capturing the nucleic acid analog to the solid support.
Alternatively, the capture process may comprise exposing the nucleic acid analog and chelatable metal ions to a solid support capable of binding the metal ions under conditions such that the metal ions become bound to the solid support and chelated by the chelating moiety of the nucleic acid analog. The metal ions can if preferred be chelated by the nucleic acid analog or to the solid support prior to the nucleic acid analog and the solid support being exposed to one another.
The solid support may comprise a chelating agent such as NTA or EDT A bound thereto chelating ions such as nickel or copper ions which are further chelatable by said nucleic acid analog.
A particularly preferred solid support is agarose gel and the solid support bearing chelatable metal ions may preferably be Ni-NTA-agarose. Conveniently, the gel may be in a column through which a solution containing the nucleic acid analog to be captured may be passed, e.g. a spin column through which said solution is centrifuged. Another preferred form of solid support is magnetic particles with a surface bearing chelatable metal ions, which may be held thereon by chelating agents as described above.
Such a method preferably comprises capturing said nucleic acid analog from a first volume of solution by a method as described, removing the solid support and captured nucleic acid analog from said solution and eluting the nucleic acid analog from the solid support in a quantity of liquid such as to produce a second volume of a solution of said nucleic acid analog which is less than said first volume of solution. The nucleic acid analog is thereby concentrated with respect to its starting solution concentration. The elution may be carried out with an excess of chelating agent such as EDTA.
A solid support having a nucleic acid analog bound thereto or capable of capturing such a nucleic acid analog by the techniques described above maybe used to capture from solution a nucleic acid of complementary sequence. A particular virtue of this technique is that one then has the option of removing the captured nucleic acid from the solid support either with or without the nucleic acid analog.
Thus by treating the system with an excess of a chelating agent such as EDTA, the chelated metal can be removed, so freeing the nucleic acid analog, and any hybridized nucleic acid. Alternatively, one may liberate the nucleic acid from the nucleic acid analog on the support by heat or other denaturing methods.
One example of such capture of a nucleic acid would be to hybridize a nucleic acid to the nucleic acid analog capture probe bearing a chelating moiety, and then to capture the resulting complex on a solid bearing metal ions.
When standard DNA probes are used in hybrid selection procedures one of the serious limitations is target sequence inaccessibility due to competing hybridization events. For instance, when targeting double-stranded PCR products the DNA probe competes with the complementary non-target PCR strand Target sequence inaccessibility can also be caused by secondary and higher order structures in the target nucleic acid. Such structures are well characterized in the case of many metabolically stable RNAs (RNA, tRNA and snRNAs). We have shown that PNA can hybridize to its complementary nucleic acid over a broad range of salt concentrations without loss of affinity and specificity. In fact the affinity of the PNA increases as the salt concentration in the buffer decreases. In theory, this is a most useful property of PNA as it allows hybridization to its target sequence under conditions of low salt that destabilizes normal nucleic acid structures. We have provided an example that this property of PNA can be used to capture a xe2x80x9cdifficultxe2x80x9d oligonucleotide in which the PNA target sequence is designed to form one side of an intra-molecular, perfectly matched 15 bp stem structure.
Methods that facilitate the rapid purification of nucleic acids from complex biological samples are important tools in both basic research and in DNA diagnostics. Compared to methods that rely on physical properties of the nucleic acids for purification, su h as density, binding to surfaces, solubility, the hybrid selection method described here offers two main advantages. Firstly, it utilizes a property that is unique to nucleic acidsxe2x80x94namely the ability to hybridize to a probe of complementary sequence. Hence, the chance of copurification of other cellular components that may prove inhibitory to downstream applications are likely to be minimal. Secondly, the method allows specific nucleic acids to be targeted thereby removing bulk DNA and RNA that may add to the generation of non-specific background in subsequent target detection procedures.
The invention includes in a third aspect thereof a labeled nucleic acid analog comprising a nucleic acid analog according to the first aspect of the invention, having chelated thereto via said chelating moiety a metal ion as label or having a labeling moiety linked thereto via a metal ion chelated by said chelating moiety.
Said metal ion is preferably a radio label such as 111indium or 99technetium or a fluorescent label such as europium or terbium.
The compounds and methods of a present invention provide a very rapid method for analyzing nucleic acids. The hybridization with the compounds of the invention can be used to define very efficient assays with a great specificity. The use of low salt conditions provides a method for analyzing even nucleic acids containing stem loop structures. It further allows the separation of nucleic acids differing by only one nucleotide. The compounds are very easy to prepare because peptide chemistry can be used to couple the complexing agent to the back bone.
Further the present compounds can be used efficiently as labeled probes in the analysis of PCR products, because they compete very efficiently with the counter strands. Further the compounds of the present invention show the superior property that also large RNAs can be captured and/or determined.
Nucleic acid analogs according to the first aspect of the invention may be prepared by first synthesizing a PNA by the solid phase techniques described in WO 92/20703 to produce a Boc-terminated PNA bound to a solid support at its carboxy end. The PNA may then be extended by removal of the Boc group to yield a starting point for a standard boc type or Fmoc type solid phase peptide synthesis adding for instance the required chelating amino acids via the linker 6-aminohexanoic acid. The protection groups may then be removed and the product may be cleaved from the resin by the Low-High TFMSA procedure. The raw product may be purified by preparative HPLC (suitable conditions being: reversed phase C18 eluting with a gradient of A: 0.1% TFA in water and B: 0.1%, 10% water, 89% acetonitrile).